Our goal is to develop small molecule inhibitors that target influenza entry. In Phase I, we identified several compounds that selectively inhibit the hemagglutinin (HA) mediated virus entry process. Three HA specific entry inhibitors (MBX494, MBX994, and MBX726) were selected for further research and development based on their antiviral potency, selectivity and chemical tractability. These influenza inhibitors display high potency (IC50 = 0.3-11 M) and favorable selectivity index values (SI>20-200) against a wide spectrum of influenza virus strains, including the high pathogenic avian influenza (HPAI) A/H5N1, 2009 pandemic influenza A/H1N1 and an oseltamivir resistant A/H1N1 strain. The inhibitors also displayed synergy with the neuraminidase inhibitor, oseltamivir. MBX494, MBX994, and MBX726 act in a group-specific manner to inhibit the H1 and H5 subtypes of group 1 HA but not the H3 or H7 subtypes of group 2 HA. Preliminary mechanism of action (MOA) studies suggest that the compounds act early during infection and inhibit the HA-mediated virus-cell membrane fusion process. Failure of the inhibitors to block influenza viruses with group 2 HA, VSV and LASV, further suggests that they do not act on cellular factors (e.g., endosomal pH, or sialic acid residues or viral/cell membrane). MBX494, MBX994, and MBX726 have aminoalkyl phenol ether, aminoacetamide sulfonamide and simple sulfonamide scaffolds respectively. Preliminary chemical optimization has already generated new active analogs, establishing the suitability of these chemical scaffolds for optimization. In Phase II, we will design, synthesize, and evaluate analogs as HA specific inhibitors. Inhibitor design will be driven by potency, target selectivity, influenza spectrum, synergistic activity with oseltamivir, ad minimal cytotoxicity. The most promising lead inhibitors will be evaluated further for favorable (i in vitro ADME properties, including stability, as well as (ii) in vivo pharmacokinetic (PK) and pharmacodynamic (PD) properties, (iii) toxicity, and (iv) efficacy in animal models to select final pre-clinical candidates. We have five specific aims. In Aim 1, we will design and synthesize analogs of the hit series to establish the SAR and improve potency. We will also use structure based drug design (SBDD) to increase the anti-influenza potency (IC50 <0.1 M) and minimize cytotoxicity (CC50 >100 M). In Aim 2, we will evaluate the analogs for potency and cytotoxicity in vitro to generate 10-20 prioritized leads. In Aim 3, we will further prioritize inhibitors by evaluating in vitro drug-like ADME properties. In Aim 4, we will analyze the MOA of anti-influenza activity. In Aim 5, we will validate the prioritized lead inhibitors for efficacy and toxcity in murine models to identify the best preclinical development candidate. Aims 1, 2, 3 and 5 constitute the preclinical candidate optimization process since together they provide the relationships between structure and activity, in vitro ADME and in vivo safety and efficacy in vivo. Understanding the MOA of the newly synthesized analogs will ensure that analogs maintain the desired mechanism and do not acquire additional or different non-specific mechanisms.